For an assay to be accurate, where the analyte concentration is read off a standard curve, the standards and patient samples must behave indentically in the assay. Perhaps one of the most critical factors between the standards and the patient samples is that the recovery of analyte from the standards must be identical to the recovery of the analyte from the patient samples or erroneous values will result. This is particularly true if the total quantity of analyte in the patient sample is being measured.
In fluorescent polarizaton assays of analytes in serum, one of the undesirable features of this methodology is the high fluorescence background of the serum. This becomes particularly troublesome when low levels of analytes like digoxin are being measured. In this case, the assay is not feasible using serum directly with a fluorescein-labeled digoxin tracer since the serum background fluorescence far exceeds the fluorescence signal emitted by the fluorescein-labeled digoxin tracer.
In order to overcome the high background fluorescence of the serum, protein-free filtrates have typically been made using perchloric or trichloroacetic acids. Although the methods are effective in producing a protein-free filtrate relatively free of background fluorescence, they suffer from other disadvantages. In the case of digoxin, relatively poor recovery is obtained in the supernatant, and in addition the recovery is dependent on the protein concentration in the sample. See for example Porter et al., Clin. Chem. 30, 1826-1829 (1984).
The use of ultrafiltration to produce protein-free filtrates is also known in the art. In such a system, protein becomes selectively partitioned into a fraction of the sample volume (retentate) while free ligand passes essentially unhindered through the membrane along with solvent into the ultrafiltrate. Systems for carrying out such ultrafiltration are commercially available. One such system is the Centrifree.TM. micropartition system sold by Amicon Corporation located in Danvers, Mass. This is basically a self-contained filter device with a filter membrane of such porosity that about 99.9% of the proteins in serum are prevented from passing through upon centrifugal filtration. The promotional literature accompanying the filters suggest they are to be used for separating "free" serum analytes, particularly drugs, from the protein bound fraction. Thus, in the absence of a dissociating agent to free drug bound to protein, only the free drug in serum would be allowed to pass through the filter and thus be measured in an assay.
Reports in the literature suggest that about 20 to 30% of digoxin in serum is bound to serum proteins. See Pribor et al., Drug Monitoring and Pharmokinetic Data, Pathotex Publishers, Park Forrest South, Ill., p. 57 (1980). Thus, if .sup.3 H-digoxin were added to human serum, about 70 to 80% of the label would be expected to be recovered in the filtrate. Using 10 normal human sera samples to which .sup.3 H-digoxin was added, followed by centrifugal filtration through the ultrafilters; it was found that 15 to 25% of the digoxin in these sera could not be recovered in the filtrate and was thus presumably protein bound.